Killing of gold nanorod-loaded human cardiac fibroblasts mediated by photo-thermal activation

Photo-thermal therapy (PTT) is a minimally invasive treatment that uses photosensitizers to convert light into heat, inducing selective cell death. Gold nanoparticles (AuNPs), thanks to tunable physical-chemical properties, are increasingly used in biomedical applications. Cardiac fibrosis is a pathogenic mechanism in several cardiovascular diseases, and is mainly driven by cardiac fibroblasts (CFs), which activate, differentiate into myofibroblasts, and enhance ECM production. Previous studies have shown partial depletion of activated CFs has beneficial effects.

To establish an innovative approach exploiting AuNP-mediated PTT for selective ablation of AuNP-loaded CFs.

Human CFs (hCFs) were treated for 72 hours with gold nanorods (AuNRs) at 50, 100, and 200 µM, cultured alone or mixed with non-treated cardiomyocytes, and irradiated with a continuous wave diode laser operating at 808 nm for 10 minutes. Cellular uptake, spectroscopy, near infrared (NIR) irradiation, heating, and cell death were assessed.

MTS assay and flow cytometry confirmed AuNR biocompatibility, with comparable viability of treated hCFs to controls (mean live cells: 85.9±2.4%). Spectroscopic analysis of treated cells revealed absorption signals associated with the AuNR longitudinal plasmon band above 800 nm, with concentration-dependent increase of intensity and redshift (λmax: 843 nm at 50 µM; 917 nm at 100 µM; 1036 nm at 200 µM). Transmission electron microscopy showed hCFs actively uptake AuNRs in endocytic-like vacuoles. Upon NIR irradiation, AuNR-hCFs exhibited a concentration-dependent temperature rise (ΔT: 12.8 °C at 0 µM; 21.5 °C at 50 µM; 22.5 °C at 100 µM; 26.2 °C at 200 µM). Flow cytometry analysis of AnnexinV/PI staining showed dose-dependent necrosis/late apoptosis (total dead cells: 26.1% at 0 µM; 54.1% at 50 µM; 59.5% at 100 µM; 76.4% at 200 µM). Selective ablation of the sole AuNR-loaded hCFs, without damaging surrounding non-loaded cells, was assessed in a 50:50 co-culture with non-treated GFP-hCFs. After irradiation, live/dead staining showed exclusive death of AuNR-hCFs, while GFP-hCFs remained viable. The minimal distance between nuclei of dead and living cells was 10.72 µm (mean distance: 35.1±14.3 µm). Selectivity was also confirmed in a more physiologically relevant model combining AuNR-hCFs and non-treated GFP-HL-1 cardiomyocytes: irradiation produced a maximum ΔT of 17.9 °C. Flow cytometry showed a strong reduction of live AuNR-hCFs (40.67±5.67%), while GFP-HL-1 viability was preserved. Necrotic and late apoptotic populations were significantly higher in AuNR-hCFs (38.1±1.8% and 20.7±4.2%) than in GFP-HL-1 (4.2±0.6% and 1.3±0.4%). n=3; p<0.001.

These findings demonstrate that hCFs can internalize AuNRs and be effectively and selectively ablated through PTT, paving the way for the development of a potential non-invasive and drug-free therapy for the ablation of detrimental cells in cardiac fibrosis.